1. Field of the Invention
This invention relates to an analysis method and apparatus utilizing attenuated total reflection, such as a surface plasmon resonance analysis method and apparatus for making an analysis of a sample by the utilization of occurrence of surface plasmon.
2. Description of the Related Art
In metals, free electrons vibrate collectively, and a compression wave referred to as a plasma wave is thereby produced. The compression wave occurring on the metal surface and having been quantized is referred to as the surface plasmon.
Various surface plasmon analysis apparatuses for analyzing characteristics of a substance to be analyzed by the utilization of a phenomenon, in which the surface plasmon is excited by a light wave, have heretofore been proposed. As one of well known surface plasmon analysis apparatuses, a surface plasmon analysis apparatus utilizing a system referred to as the Kretschman arrangement may be mentioned. The surface plasmon analysis apparatus utilizing the system referred to as the Kretschman arrangement is described in, for example, Japanese Unexamined Patent Publication No. 6(1994)-167443.
Basically, the surface plasmon analysis apparatus utilizing the system referred to as the Kretschman arrangement comprises (i) a dielectric material block having, for example, a prism-like shape, (ii) a metal film, which is formed on one surface of the dielectric material block and is brought into contact with a substance to be analyzed, such as a liquid sample, (iii) a light source for producing a light beam, (iv) an optical system for irradiating the light beam to the dielectric material block at various different incidence angles such that a total reflection condition may be satisfied at an interface between the dielectric material block and the metal film, and (v) a photo detecting means for detecting the intensity of the light beam, which has been totally reflected from the interface described above, and thereby detecting the state of surface plasmon resonance, i.e. the state of attenuated total reflection.
In order for the various different incidence angles described above to be obtained, a light beam having a comparatively small beam diameter may be caused to impinge upon the aforesaid interface with the incidence angle being altered. Alternatively, a light beam having a comparatively large beam diameter may be caused to impinge upon the aforesaid interface in a state of converged light or in a state of a divergent light, such that the light beam may contain components, which impinge at various different incidence angles upon the interface. In the former case, the reflected light beam, which is reflected from the interface with its reflection angle altering in accordance with the alteration of the incidence angle of the incident light beam, may be detected with a small photodetector, which moves by being interlocked with the alteration of the reflection angle, or may be detected with an area sensor extending in the direction of alteration of the reflection angle. In the latter case, the light beam may be detected with an area sensor extending in a direction such that the area sensor is capable of receiving all of the light beam components having been reflected from the interface at various different reflection angles.
With the surface plasmon analysis apparatus having the constitution described above, in cases where the light beam impinges at a specific incidence angle, which is not smaller than the total reflection angle, upon the metal film, an evanescent wave having an electric field distribution occurs in the substance to be analyzed, which is in contact with the metal film, and the surface plasmon is excited by the evanescent wave and at the interface between the metal film and the substance to be analyzed. In cases where the wave vector of the evanescent wave coincides with the wave vector of the surface plasmon, and wave number matching is thus obtained, the evanescent wave and the surface plasmon resonate, and energy of the light transfers to the surface plasmon. As a result, the intensity of the reflected light beam, which is totally reflected from the interface between the dielectric material block and the metal film, becomes markedly low. Ordinarily, the lowering of the intensity of the reflected light beam is detected as a dark line by the photo detecting means described above. The resonance described above occurs only in cases where the incident light beam is P-polarized light. Therefore, it is necessary for the incident light beam to be set previously so as to impinge upon the aforesaid metal film as the P-polarized light.
In cases where the wave number of the surface plasmon is found from the incidence angle at which the attenuated total reflection (ATR) occurs, i.e. from an attenuated total reflection angle (ATR angle) θSP, a dielectric constant of the substance to be analyzed is capable of being calculated. Specifically, the formula shown below obtains.
            K      SP        ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              wherein KSP represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the light velocity in a vacuum, εm represents the dielectric constant of the metal, and εs represents the dielectric constant of the substance to be analyzed.
Specifically, in cases where the ATR angle θSP, which is the incidence angle associated with the lowering of the intensity of the reflected light described above, is found, the dielectric constant εs of the substance to be analyzed is capable of being calculated. Therefore, the characteristics with regard to the refractive index of the substance to be analyzed are capable of being calculated.
Such that the ATR angle θSP may be measured accurately and with a wide dynamic range, a technique has been proposed, in which array-like photo detecting means is utilized in the aforesaid type of the surface plasmon analysis apparatus. (The proposed technique for utilizing the array-like photo detecting means is described in, for example, U.S. Pat. No. 6,577,396.) The array-like photo detecting means comprises a plurality of light receiving devices arrayed in a predetermined direction. The array-like photo detecting means is located in an orientation such that each of the light receiving devices is capable of receiving one of components of the light beam, which components have been totally reflected at various different reflection angles from the interface described above.
In such cases, the surface plasmon analysis apparatus is often provided with differentiation means for performing differentiation of signal components of a photo detection signal, each of which signal components is outputted from one of the light receiving devices of the aforesaid array-like photo detecting means, with respect to the array direction of the light receiving devices. Also, the characteristics with regard to the refractive index of the substance to be analyzed are calculated in accordance with differentiation values, which are outputted by the differentiation means.
As a similar analysis apparatus utilizing the attenuated total reflection (ATR), a leaky mode analysis apparatus has heretofore been known. (The leaky mode analysis apparatus is described in, for example, “Surface Refracto-sensor using Evanescent Waves: Principles and Instrumentations” Takayuki Okamura, Spectrum Researches, Vol. 47, No. 1, pp. 19-28, 1998.) Basically, the leaky mode analysis apparatus comprises (i) a dielectric material block having, for example, a prism-like shape, (ii) a cladding layer, which is formed on one surface of the dielectric material block, (iii) an optical waveguide layer, which is formed on the cladding layer and is brought into contact with a liquid sample, (iv) a light source for producing a light beam, (v) an optical system for irradiating the light beam to the dielectric material block at various different incidence angles such that a total reflection condition may be satisfied at an interface between the dielectric material block and the cladding layer, and (vi) a photo detecting means for detecting the intensity of the light beam, which has been totally reflected from the interface described above, and thereby detecting the state of excitation of a guided mode, i.e. the state of attenuated total reflection.
With the leaky mode analysis apparatus having the constitution described above, in cases where the light beam impinges at an incidence angle, which is not smaller than the total reflection angle, upon the cladding layer via the dielectric material block, only the light having a certain specific wave number, which light has impinged at a specific incidence angle upon the cladding layer, is propagated in the guided mode in the optical waveguide layer after passing through the cladding layer. In cases where the guided mode is thus excited, approximately all of the incident light is taken into the optical waveguide layer. Therefore, in such cases, the attenuated total reflection occurs, and the intensity of the light totally reflected from the aforesaid interface becomes markedly low. Also, the wave number of the guided optical wave depends upon the refractive index of the substance to be analyzed, which is located on the optical waveguide layer. Therefore, in cases where the aforesaid specific incidence angle, which is associated with the occurrence of the attenuated total reflection, is detected, the refractive index of the substance to be analyzed and characteristics of the substance to be analyzed with regard to the refractive index of the substance to be analyzed are capable of being analyzed.
In the leaky mode analysis apparatus, the array-like photo detecting means described above may be utilized in order to detect the position of the dark line occurring in the reflected light due to the attenuated total reflection. Also, the differentiation means described above is often utilized together with the array-like photo detecting means.
In the fields of pharmaceutical research, and the like, the surface plasmon analysis apparatus and the leaky mode analysis apparatus described above are often utilized for random screening for finding out a specific substance, which is capable of undergoing the binding with a desired sensing substance. In such cases, the sensing substance acting as the substance to be analyzed is fixed to the aforesaid thin film layer (the metal film in the cases of the surface plasmon analysis apparatus, or the combination of the cladding layer and the optical waveguide layer in the cases of the leaky mode analysis apparatus), and a liquid sample containing a test body in a solvent is introduced on the sensing substance. Also, at each of stages after the passage of predetermined periods of time, the aforesaid ATR angle θSP is measured.
In cases where the test body contained in the liquid sample is a substance capable of undergoing the binding with the sensing substance, the refractive index of the sensing substance alters with the passage of time. Therefore, the aforesaid ATR angle θSP is measured at each of stages after the passage of predetermined periods of time, and a judgment is made as to whether an alteration of the ATR angle θSP has been or has not been occurred. In this manner, the state of the binding of the test body with the sensing substance is capable of being detected, and a judgment is capable of being made in accordance with the result of the detection and as to whether the test body is or is not the specific substance capable of undergoing the binding with the sensing substance. Examples of the combinations of the specific substances and the sensing substances include the combination of an antigen and an antibody and the combination of an antibody and a different antibody. Specifically, for example, a rabbit anti-human IgG antibody may be fixed as the sensing substance to the surface of the thin film layer, and a human IgG antibody may be employed as the specific substance.
In order for the state of the binding of the test body with the sensing substance to be detected, the ATR angle θSP itself need not necessarily be detected. Alternatively, for example, the liquid sample may be introduced on the sensing substance, and thereafter the quantity of the alteration of the ATR angle θSP may be measured. Also, the state of the binding of the test body with the sensing substance may be detected in accordance with the quantity of the alteration of the ATR angle θSP. In cases where the array-like photo detecting means and the differentiation means described above are utilized in the analysis apparatus utilizing attenuated total reflection, since the quantity of the alteration of the differentiation value reflects the quantity of the alteration of the ATR angle θSP, the state of the binding of the test body with the sensing substance is capable of being detected in accordance with the quantity of the alteration of the differentiation value.
In the analysis method and apparatus utilizing attenuated total reflection as described above, a cup-shaped or laboratory dish-shaped analysis chip, in which the sensing substance has been fixed to the thin film layer having been formed previously on a bottom surface, is prepared. Also, the liquid sample containing the test body in the solvent is introduced into the analysis chip, and the quantity of the alteration of the ATR angle θSP is measured.
In cases where the liquid sample is introduced into the analysis chip, and the sensing substance and the test body are bound to each other, the refractive index of the sensing substance alters, and the ATR angle θSP alters. Therefore, at the time at which a predetermined period of time has elapsed after the measurement has been begun, the quantity of the alteration of the ATR angle θSP occurring after the measurement has been begun may be calculated. In this manner, a judgment is capable of being made as to whether the test body is or is not a substance capable of undergoing the binding with the sensing substance. Also, in cases where it has been judged that the test body is a substance capable of undergoing the binding with the sensing substance, the state of the binding of the test body with the sensing substance, or the like, is capable of being analyzed.
As described above, the array-like photo detecting means, which is described in, for example, U.S. Pat. No. 6,577,396 and which is utilized for the enhancement of sensitivity comprises the plurality of the light receiving devices arrayed in the predetermined direction. The array-like photo detecting means is located in the orientation such that each of the light receiving devices is capable of receiving one of the components of the light beam, which components have been totally reflected at various different reflection angles from the interface described above. The components of the light beam, which components are taken in a beam width direction perpendicular to the reflection angle direction and with respect to each of the various different reflection angles, are received by one light receiving device. Therefore, the output obtained from each of the light receiving devices contains all of the information with respect to the beam width direction. However, in cases where the surface of the thin film layer of the analysis chip is not uniform, in cases where the sensing substance is not uniform, or in cases where staining occurs at part of the optical system, the problems often occur in that part of the dark line is deformed (for example, part of the dark line becomes dull). Therefore, with the conventional analysis apparatus provided with the array-like photo detecting means, in cases where part of the dark line is deformed due to defects, such as non-uniformity, staining, and the like, of part of the analysis chip or part of the optical system, a measurement result containing adverse effects of the deformation of part of the dark line is obtained. Accordingly, with the conventional analysis apparatus provided with the array-like photo detecting means, there is the possibility that the detection sensitivity will become low.